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Assessment of the roles of mitogen-activated protein kinase and phosphatidyl inositol 3-kinase/protein kinase B pathways in the basic fibroblast growth factor regulation of Sertoli cell function

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Assessment of the roles of mitogen-activated protein kinase and phosphatidyl inositol 3-kinase/protein kinase B pathways in the basic fibroblast growth factor regulation of Sertoli cell function
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  Assessment of the roles of mitogen-activated proteinkinase and phosphatidyl inositol 3-kinase/proteinkinase B pathways in the basic fibroblast growthfactor regulation of Sertoli cell function M F Riera, S B Meroni, E H Pellizzari  and  S B Cigorraga Centro de Investigaciones Endocrinológicas (CEDIE), Hospital de Niños ‘Ricardo Gutiérrez’, Gallo 1330, (1425) Buenos Aires, Argentina(Requests for offprints should be addressed to S B Cigorraga; Email: scigorraga@cedie.org.ar) Abstract Basic fibroblast growth factor (bFGF) belongs to the large set of intratesticular regulators that providethe fine tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of thepresent study was to determine the participation of mitogen-activated protein kinase (MAPK) andphosphatidyl inositol 3-kinase/protein kinase B (PI3K/PKB) pathways in bFGF regulation of Sertoli cellfunction. Twenty-day-old rat Sertoli cell cultures were used. Stimulation of the cultures with bFGF showeda time-dependent increment in phosphorylated MAPK and PKB levels that reached maximal values in5-min incubations. MAPK kinase inhibitors U0126 (U) and PD98059 (PD) and a PI3K inhibitor wortmannin(W) were able to block the stimulatory effects of bFGF on phosphorylated MAPK and PKB levelsrespectively. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF oftwo well-known Sertoli cell-differentiated functions, lactate and transferrin production, was next explored.As for lactate production, PD and W did not modify the ability of bFGF to stimulate lactate production.However, a combination of PD and W partially impaired the increase in lactate production elicited bybFGF. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF ofglucose uptake and lactate dehydrogenase (LDH) activity was also analysed. In this respect, it wasobserved that W markedly decreased basal and bFGF-stimulated glucose uptake and that U and PD didnot modify it. On the other hand, U and PD decreased the stimulation of LDH activity by bFGF whereasW did not modify it. As for transferrin production, while both MAPK kinase inhibitors partially decreasedthe ability of bFGF to stimulate transferrin secretion, the PI3K inhibitor did not modify it. In summary, theresults demonstrated that bFGF stimulates MAPK- and PI3K/PKB-dependent pathways in rat Sertolicells. Moreover, these results showed that while bFGF utilizes the MAPK pathway to regulate transferrinproduction and LDH activity, it uses the PI3K/PKB pathway to regulate glucose transport into the cell. Journal of Molecular Endocrinology   (2003)  31,  279–289 Introduction  A growing body of evidence suggests that basicfibroblast growth factor (bFGF) belongs to the largeset of intratesticular regulators that provide the finetuning of cellular processes implicated in themaintenance of spermatogenesis. bFGF exerts itse ff   ects by binding to receptors which belong to thetyrosine kinase family. Following ligand binding and dimerization, the receptors become capable of phosphorylating specific tyrosine residues on theirown cytoplasmic tails and on each other’s(Lemmon & Schlessinger 1994). Phosphorylatedtyrosine residues, in turn, recruit other signaling molecules to the activated receptors and propagatethe signal through several possible transductionpathways (Pawson 1995). Recently, Ong   et al. (2001) have shown that, with the participation of di ff   erent docking proteins, bFGF elicits bifurcating signals to activate the mitogen-activated proteinkinase (MAPK) and phosphatidyl inositol 3-kinase/protein kinase B (PI3K/PKB) pathways simul-taneously in several cell lines. However, theactivation of these signaling cascades may vary in 279 Journal of Molecular Endocrinology   (2003)  31,  279–2890952–5041/03/031–279 © 2003 Society for Endocrinology  Printed in Great Britain  Online version via http://www.endocrinology.org  di ff   erent cell types. For example, the PI3K/PKBpathway is not triggered by bFGF in smoothmuscle cells although it is an essential pathway forthe maintenance of the di ff   erentiated phenotype of these cells (Hayashi  et al.  1999). This fact probablyreflects di ff   erences in the internal machinery towhich the specific receptors are coupled in thedi ff   erent cell types.bFGF has been found to modulate variousnon-mitogenic biological processes in a wide rangeof tissues and organs, including the testis, wherebFGF has been shown to be produced (Ueno  et al. 1987). Germ cells are a potential source of thispeptide although other cells in the testis alsoexpress it (Mullaney & Skinner 1992, Han  et al. 1993). bFGF receptors are constitutively expressedin Sertoli cell cultures (Le Magueresse-Battistoni et al.  1994) and it has been shown that bFGFmodulates, among other responses, the numberof follicle-stimulating hormone (FSH) receptors(Jaillard  et al.  1987), transferrin, estradiol andlactate secretion (Han  et al.  1993, Schteingart  et al. 1999), glucose uptake, lactate dehydrogenase(LDH) activity and the levels of glucose transporter1 and LDH A mRNA (Riera  et al.  2002).Thus far, the signal transduction pathways thatmay be activated by bFGF in Sertoli cells have notbeen analyzed. Furthermore, no studies areavailable at present on the possible relationshipbetween activated signaling cascades and specificbiological responses in rat Sertoli cells. Therefore,the aim of the present study was to determine (a)whether bFGF is able to stimulate MAPK- andPI3K/PKB-signaling pathways in Sertoli cells and(b) to what extent these signaling pathwaysparticipate in bFGF regulation of Sertoli cellfunction. Materials and methods Materials Human recombinant bFGF and tissue culturemedia were purchased from GIBCO BRL (LifeTechnologies Ltd, Rockville, MD, USA).Wortmannin (W), U0126 (U) and PD98059 (PD)were purchased from Biomol (Plymouth Meeting,PA, USA). [2,6- 3 H]-2-deoxy-  -glucose (2-DOG)was purchased from NEN (Boston, MA, USA). Allother drugs and reagents were purchased fromSigma Chemical Co. (St Louis, MO, USA). Sertoli cell isolation and culture Sertoli cells from 20-day-old Sprague–Dawley ratswere isolated as previously described (Meroni  et al. 1999). Briefly, decapsulated testes were digestedwith 0·1% collagenase and 0·006% soybean trypsininhibitor in Hanks’ balanced salt solution for 5 minat room temperature. Seminiferous tubules weresaved, cut and submitted to 1 M glycine–2 mMEDTA (pH 7·4) treatment to remove peritubularcells. The washed tubular pellet was then digestedagain with collagenase for 10 min at roomtemperature to remove germinal cells. The Sertolicell suspension, collected by sedimentation, wasresuspended in culture medium which consisted of a 1:1 mixture of Ham’s F12 and Dulbecco’smodified Eagle’s medium, supplemented with20 mM HEPES, 100 IU/ml penicillin, 2·5 µg/mlamphotericin B, 1·2 mg/ml sodium bicarbonate,10 µg/ml transferrin, 5 µg/ml insulin, 5 µg/ml vitamin E and 4 ng/ml hydrocortisone. Sertoli cellswere cultured in 6- or 24-multiwell plates (5 µg DNA/cm 2  ) at 34   C in a mixture of 5% CO 2 :95%air.No myoid cell contamination was revealed inthe cultures when an immunoperoxidase tech-nique was applied to Sertoli cell cultures using aspecific antiserum to smooth muscle    actin.Remaining cell contaminants were of germ cellsrcin and this contamination was below 5% after48 h in culture as examined by phase contrastmicroscopy. Culture conditions Sertoli cells were allowed to attach for 48 h in thepresence of insulin and medium was replaced atthis time with fresh medium without insulin.Stimulation with bFGF was performed on day 3in the presence or absence of MAPK kinase andPI3K inhibitors (U, PD and W) as indicated inthe figure legends. In all cases the inhibitorswere added 15 min prior to the addition of bFGF. The 72-h conditioned media obtained onday 6 was used to evaluate transferrin and lactatelevels. Cells harvested on day 6 were used todetermine LDH activity. For 2-DOG uptakestudies, cells cultured for 4 days under basalconditions pretreated for 2 h with bFGF in thepresence or absence of the above-mentionedinhibitors were used. M F RIERA  and others  · bFGF regulates MAPK and PI3K/PKB pathways  280 www.endocrinology.org Journal of Molecular Endocrinology   (2003)  31,  279–289  Cell extracts and Western blot analysis Cells harvested on day 6 and cultured on 6-wellplates, pretreated as indicated in the figure legends,were used for Western blot analysis. Cells werewashed once with phosphate-bu ff   ered saline (PBS)at room temperature. Then, 200 µl PBS containing 20 µl of a protease inhibitor cocktail from Sigma(P-8340) and 2 mM phenylmethylsulfonyl fluoridewere added to the cells. Cells were then placed onice and disrupted by ultrasonic irradiation. A 200 µl volume of 2  Laemmli bu ff   er (4% (w/v) SDS,20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol,0·004% (w/v) bromophenol blue and 0·125 MTris–HCl, pH 6·8) was added and thoroughlymixed (Laemmli 1970). Samples were immersedin a boiling water bath for 5 min and then  im-mediately settled on ice. Proteins were resolved in10% SDS-PAGE (10% acrylamide/bisacrylamidefor the resolving gel and 4·3% acrylamide/bisacrylamide for the stacking gel) in a Mini Protean3 Cell (Bio-Rad, Hercules, CA, USA). AfterSDS-PAGE, gels were equilibrated in transferbu ff  er for 10 min and electrotransferred at 100 V for60 min onto polyvinylidene difluoride membranes(Hybond-P; Amersham Pharmacia Biotech, Amersham, Bucks, UK) using a mini trans-blot cell(Bio-Rad). Membranes were probed with com-mercial kits (phosphoplus Akt Ser 473 antibody kitand phosphoplus p44/42 MAP kinase (Thr202/Tyr204) antibody kit; New England Biolabs Inc.,Beverley, MA, USA) that allow specific recognitionof both total (T-PKB and T-MAPK) and phos-phorylated (P-PKB and P-MAPK) PKB and MAPK.The intensities of the autoradiographic bands wereestimated by densitometric scanning using NIHImage (Scion Corporation, Frederick, MD, USA)software. Transferrin determination Rat transferrin was measured by radioimmuno-assay (RIA) as described by Handelsman  et al. (1989). A polyclonal antibody raised againstrat transferrin in rabbits was used (Cappel Laboratories, Cochranville, PA, USA). Thecross-reactivity of human transferrin in thisassay is less than 0·003%. This RIA has asensitivity of 3 ng/tube and intra- and interassaycoe ffi cients of variation are 7% and 16%respectively. Measurement of 2-DOG Glucose transport was studied using the uptake of the labeled non-metabolizable glucose analogue2-DOG. Cells were washed three times withglucose-free PBS at room temperature. Sertoli cellswere then incubated at 34   C in 0·5 ml glucose-free PBS containing [2,6- 3 H]-2-DOG (0·5 µCi/ml)for 30 min. Unspecific uptake was determined inincubations performed in the presence of a10 000-fold higher concentration of unlabeled2-DOG. At the end of the incubation period, disheswere placed on ice and washed extensively withice-cold PBS until no radioactivity was present inthe washings. Cells were then dissolved with 0·5 Msodium hydroxide and 0·4% sodium deoxycholateand counted in a liquid scintillation spectro-photometer. Parallel cultures receiving identicaltreatments to those performed before the glucoseuptake assay were destined for DNA deter-minations. Results are expressed on a per µg DNAbasis. LDH activity measurement  After incubation of Sertoli cells in the absence orpresence of the di ff   erent stimuli, culture mediumwas discarded and cells were disrupted byultrasonic irradiation in NaCl (0·9%) and centri-fuged (15 800  g  , 10 min). The supernatant wasused to measure total LDH activity. Total LDHactivity was determined by a routinely usedspectrophotometric method (Randox Laboratories,Crumlin, UK). Lactate determination Lactate was measured by a standard methodinvolving conversion of NAD + to NADH deter-mined as the rate of increase of absorbance at340 nm. A commercial kit from Sigma-Aldrich(St Louis, MO, USA) was used. Cell viability test  A cell viability test was performed in cells culturedon 96-well plates and treated for 72 h with U, PD,W or a combination of both PD and W. Acommercial kit (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay; PromegaCorporation, Madison, WI, USA) was used. bFGF regulates MAPK and PI3K/PKB pathways   ·  M F RIERA  and others 281 www.endocrinology.org  Journal of Molecular Endocrinology   (2003)  31,  279–289  Other assays DNA was determined by the method of Labarca &Paigen (1980). Statistical analysis To analyze data from glucose uptake studies,transferrin and lactate production and LDHactivity, one-way analysis of variance followed bythe Tukey–Kramer test for multiple comparisonsusing the GB-STAT version 4·0 statistical program(Dynamic Microsystems Inc., Silver Spring, MD,USA) was performed. Probabilities  , 0·05 wereconsidered statistically significant. Results bFGF increases phosphorylated MAPK andPKB levels in rat Sertoli cells In order to evaluate a possible e ff   ect of bFGF onthe levels of P-MAPK and P-PKB, Sertoli cellcultures were stimulated for variable periods of time (5, 15 and 30 min) with a dose of bFGF(30 ng/ml) that has been shown to elicit maximalbiological responses (Riera  et al.  2002). Stimulationof the cultures with bFGF produced a time-dependent increment in P-MAPK and P-PKBlevels that reached maximal values in 5-minincubations (Fig. 1A and B respectively). WhileP-PKB levels decreased in incubations longerthan 5 min, P-MAPK levels remained elevated in60- and 90-min incubations (five- and fourfoldrespectively, data not shown).We next examined whether MAPK kinaseinhibitors, U and PD, and a PI3K inhibitor, W,were able to block the stimulatory e ff   ects of bFGFon P-MAPK and P-PKB levels. Cells werepreincubated for 15 min with the inhibitors andthen stimulated with bFGF for 5 min. Figure 2Ashows that U (left panel) and PD (middle panel)dose-dependently decreased the ability of bFGF toincrease the levels of P-MAPK and that W (right Figure 1  Effect of bFGF on P-MAPK and P-PKB levels in rat Sertoli cells. Sertoli cells were stimulatedfor variable periods of time (5, 15 and 30 min) with 30 ng/ml bFGF. Cell extracts were prepared at thedesignated intervals and utilized for Western blot analysis using antibodies specific for (A) T-MAPK orP-MAPK and (B) T-PKB or P-PKB. The upper panels show a representative experiment out of four.The lower panels show pooled data of four independent experiments indicating the fold increase inphosphorylation (ratio of P-MAPK to T-MAPK and of P-PKB to T-PKB in each sample) relative tobasal. Results are expressed as means± S.D. M F RIERA  and others  · bFGF regulates MAPK and PI3K/PKB pathways  282 www.endocrinology.org Journal of Molecular Endocrinology   (2003)  31,  279–289  Figure 2  Effect of MAPK kinase and PI3K inhibitors on bFGF-stimulated levels of P-MAPK and P-PKB in rat Sertolicells. (A) Sertoli cells preincubated or not for 15 min with U (0·1 and 1 µM), PD (1 and 10 µM) and W (0·1 µM) werestimulated for 5 min with 30 ng/ml bFGF. Cell extracts were prepared and utilized for Western blot analysis usingantibodies specific for T- MAPK or P-MAPK. The upper panels show a representative experiment out of three. Thelower panels show pooled data of three independent experiments indicating the fold increase in phosphorylation(ratio of P-MAPK to T-MAPK in each sample) relative to basal. Results are expressed as means± S.D.  (B) Sertolicells preincubated or not for 15 min with W (0·01 and 0·1 µM), U (1 µM) and PD (10 µM) were stimulated for 5 minwith 30 ng/ml bFGF. Cell extracts were prepared and utilized for Western blot analysis using antibodies specific forT- PKB or P-PKB. The upper panels show a representative experiment out of three. The lower panels show pooleddata of three independent experiments indicating the fold increase in phosphorylation (ratio of P-PKB to T-PKB ineach sample) relative to basal. Results are expressed as means± S.D. bFGF regulates MAPK and PI3K/PKB pathways   ·  M F RIERA  and others 283 www.endocrinology.org  Journal of Molecular Endocrinology   (2003)  31,  279–289
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